(i) Field of the Invention
The present invention pertains to the therapeutic use of a peptide molecule derived from maturation products of SMR1 (Submandibular rat protein 1).
(ii) Description of the Related Art
The intracellular or systemic hydro-mineral imbalance of the body of a mammal, and more specifically of the human body is the cause of multiple pathologies affecting the metabolism and the physiological behavior of diverse organs and tissues, such as bone, kidney, parathyroid, pancreas, intestine, the glandular mucosa of the stomach or the prostate as well as salivary glands.
In the body of a mammal, the maintenance of the transmembrane potassium/sodium and magnesium/calcium ratios is critically important in the control of cell excitation and the regulation of many aspects of intracellular metabolism. The most active tissues such as nerve, liver and muscle have a higher ratio of potassium/sodium and magnesium/calcium than inactive tissues such as skin and erythrocytes. In addition, the most active tissues have a higher phosphorus content than inactive tissues, in keeping with the role of phosphate esters in cellular energy metabolism.
An adult human contains approximately 1,000 g of calcium (Krane et al., 1970). Some 99% of this calcium is in the skeleton in the form of hydroxyapatite, and 1% is contained in the extracellular fluids and soft tissues. About 1% of the skeletal content of calcium is freely exchangeable with the extracellular fluids. Although small as a percentage of skeletal content, this exchangeable pool is approximately equal to the total content of calcium in the extracellular fluids and soft tissues, and serves as an important buffer or storehouse of calcium. Thus, calcium plays two predominant physiological roles in the organism. In bone, calcium salts provide the structural integrity of the skeleton. In the extracellular fluids and in the cytosol, the concentration of calcium ions is critically important in the maintenance and control of a number of biochemical processes, and the concentrations of Ca2+ in both compartments are maintained with great constancy (Broadus, 1993). Other important mineral ions such as sodium, magnesium or phosphorus are deeply involved in the mineral ion balance necessary for a good intra- and extra-cellular metabolism. The term mineral ion balance refers to the state of mineral homeostasis in the organism vis-à-vis the environment. In zero balance, mineral intake and accretion exactly match mineral losses; in positive balance, mineral intake and accretion exceed mineral losses, and in negative balance, mineral losses exceed mineral intake and accretion. Under normal circumstances net calcium absorption provides a surplus of calcium that considerably exceeds systemic requirements.
The extracellular pool of orthophosphate (about 550 mg in human) is in dynamic equilibrium with phosphorus entry and exit via the intestine, bone, kidney and soft tissues. In zero balance, fractional net phosphorus absorption is about two-thirds of phosphorus intake. This amount represents a vast excess over systemic requirements and is quantitatively excreted into the urine.
The extracellular pool of magnesium (about 250 mg in human) is in bidirectional equilibrium with magnesium fluxes across the intestine, bone, kidney and soft tissues. In zero balance, the magnesium derived from the net intestinal absorption (about 100 mg/day in human) represents a systemic surplus and is quantitatively excreted.
Two organs are mainly involved in the absorption and excretion of the different mineral ions of the body: 1) Hormonal and/or intrinsic mechanisms of mineral ion absorption in the intestine provide the body with a mineral supply that exceeds systemic mineral needs by a considerable measure; 2) the renal tubule plays the dominant quantitative role in maintaining normal mineral homeostasis.
Few endogenously produced metabolites have already been shown to participate actively in the maintenance of the mineral ion balance within the body.
The 1,25-dihydroxyvitamin D (also named calcitriol) is the only recognized hormonal stimulus of active intestinal calcium absorption that occurs principally in the duodenum and the jujenum (Lemann Jr J., 1993). As a consequence, reduced net intestinal calcium absorption occurs when either dietary calcium intake is limited, when serum 1,25-dihydroxyvitamin D concentrations are low or when the intestine is unresponsive to this hormone. In contrast, increased intestinal calcium absorption occurs when serum 1,25-dihydroxyvitamin D concentrations are high. Thus defects in the regulation of the 1,25-dihydroxyvitamin D concentration in the serum can cause major disorders reducing or enhancing intestinal calcium absorption and lead to a pathological state. The 1,25-dihydroxyvitamin D also influences the body intake of phosphate.
A second endogenous factor involved in the mineral ion balance is the parathyroid hormone (PTH). Parathyroid hormone regulates the level of calcium and phosphate in blood by modulating the activity of specific cells in bone and kidney. These actions serve to: 1) stimulate reabsorption of calcium and phosphate from bone; 2) stimulate reabsorption of calcium and inhibit reabsorption of phosphate from glomerular filtrate; and 3) stimulate the renal synthesis of 1,25-dihydroxyvitamin D thereby increasing intestinal absorption of calcium and phosphate.
A third endogenous factor intervening in the mineral ion balance is calcitonin. Calcitonin (CT) is a 32-amino-acid peptide that is secreted primarily by thyroidal C-cells (Deftos, 1993). Its main biological effect is to inhibit osteoclastic bone resorption. This property has led to CT""s use for disorders characterized by increased bone resorption, such as Paget""s disease, osteoporosis and for the hypercalcemia of malignancy. The secretion of CT is regulated acutely by blood calcium and chronically by gender and perhaps age. Calcitonin is metabolized by the kidney and the liver. The amino acid sequence of CT is widely conserved through the evolution, from fish to mammals.
Defects in the mineral ion balance are the cause of multiple disorders affecting either the bone, the kidney, the intestine, the pancreas, the dental tissues (enamel and ivory), or the stomach mucosa.
A mineral ion imbalance affects the bone remodeling capacity causing disorders such as osteoporosis or affects the bone resorption capacity such as in the hyper-parathyroidism disease. The bone remodeling system has been characterized in numerous publications in the recent past (Parfitt, 1986). Bone remodeling occurs on trabecular and Haversian bone surfaces. The first step is activation of osteoclast precursors to form osteoclasts that then begin to excavate a cavity on a surface. After removal of bone tissues (about 0.05 mm 3), the site remains quiescent for a short time, following which activation of osteoblast precursors occurs at the site and the excavation is refilled. This process serves several functions, among them the removal of aged, microdamaged bone tissue and rearrangement of the bone architecture to meet the needs of mechanical support. With normal daily use of the skeleton, bone loss abnormal accumulation of microdamage, or errors in geometry can come about only through defects in this system, for example a defect in the mineral ion balance. Osteoporosis is a major public health concern and there is consequently a great need for new therapeutical molecules that will be able to regulate the mineral ion balance in the body and, if possible, more efficient and more selective (target specific) than the molecules presently used in therapeutic, such as oestrogen and calcitonin. Other bone absorption or resorption diseases may be caused by defects in the renal or gastrointestinal mineral ion metabolism, such as renal osteodystrophy or even caused by a pancreatic insufficiency.
Primary hyperparathyroidism is a very common cause of hypercalcemia, with estimates of incidence as high as 1 in 500 to 1 in 1000 (Bilezikian, 1990). Hyperparathyroism is a hypercalcemia occurring in association with elevated levels of parathyroid hormone, often caused by a benign, solitary adenoma.
Hypercalcemia may be the result of diverse other disorders such as familial hyperparathyroid syndromes (Szabo J. et al., 1993), familial hypocalciuric hypercalcemia (Marx S. J., 1993), hypercalcemia due to malignancy states (Stewart A. F., 1993; Mundy G. R., 1993) or due to granuloma-forming disorders (Adams, J. S., 1993).
On the order hand, defects in the mineral ion balance can result in hypocalcemia which is encountered in diseases associated with a low serum albumin concentration or also with idiopathic hypoparathyroidism. Hypocalcemia itself is sometimes due to hypomagnesemia or hyperphosphatemia, or to an impaired secretion of the parathyroid hormone (Shane E., 1993) or also to vitamin D disorders (Insogna K. L., 1993).
The dental tissues may also be affected in the case of a defect in the mineralization and formation of the dental ivory or enamel. Pancreas is also an organ very sensitive to a defect in mineral ion equilibrium, which is able to cause an inflammation named pancreatitis. Even the submandibular gland may be affected, causing a pathological state of lithiasis which is associated with calcium deposits. The kidney may also be affected, resulting in the development of nephrolithiasis.
In the same way, aluminum accumulation in uremic patients is associated with bone disease, which is characterized by reduced bone formation leading to osteodystrophy (Sherrard et al., 1988).
Two other disorders of public health concern are respectively a hypercalcemia resulting from medications (Stewart, 1993) and the parathyroid hormone resistance syndromes (Levine, 1993).
Due to the multiple disorders caused by a decrease or an increase of the mineral ion metabolism (principally calcium, magnesium, phophorus or aluminum ions) and the very small number of molecules that are, to date, of therapeutic value in the prevention or in the treatment of the above-described pathological states, there exists a great public need for new active molecules that are able to regulate the mineral ion concentrations within the body.
The inventors have previously characterized a new rat submandibular gland protein, named SMR1 (submandibular rat 1 protein), which has the structure of a prohormone and whose synthesis is under androgen control (Rosinsky-Chupin et al., 1988 and PCT Patent Application No. WO 90/03981) The gene encoding SMR1 belongs to a new multigene family, the VCS family, which has been localized to chromosome 14, bands p21-p22 (Courty et al., 1996; Rosinsky-Chupin et al., 1995) and for which human gene counterpart has been characterized. The gene has a similar organization to a number of hormone precursor genes (Rosinsky-Chupin et al., 1990). SMR1 mRNA is expressed in a highly tissue-; age- and sex-specific manner in the acinar cells of the male rat submaxillary gland (SMG) and in the prostate (Rosinsky-Chupin et al., 1993).
It has been described that, in vivo, SMR1 is selectively processed at pairs of basic amino acid sites in a tissue- and sex-specific manner to give rise to mature peptide products, in a manner similar to the maturation pathway of peptide-hormone precursors (Rougeot et al., 1994). Generally, this selective proteolytic fragmentation has been shown to be critical for the generation and the regulation of biologically active peptides (Lindberg et al., 1991; Steiner et al., 1992). The biosynthesis of the peptides generated from SMR1 or from its human counterpart by cleavage at pairs of arginine residues e.g. the undecapeptide: VRGPRRQHNPR (SEQ ID NO:7); the hexapeptide: RQHNPR (SEQ ID NO:12); and the pentapeptide: QHNPR (SEQ ID NO:1), is subject to distinct regulatory pathways depending on 1) the organ: SMG and prostate, 2) the developmental stage: from 6 weeks postnatal, 3) the sex: predominantly in the male, and 4) gonad hormones: the androgens. Furthermore, in vivo, the mature peptides which accumulate in the male rat SMG, are exported into the extracellular space in response to a specific external stimulus and, in this way are transported within the salivary and blood fluids (Rougeot et al., 1993). The fact that these peptides are mainly produced in postpubescent male rats and are secreted into the saliva and blood under stimulated conditions, led one to postulate that they have a local and systemic physiological role in mediating some male-specific behavioral characteristics but this role was totally unknown.
The inventors have now discovered that the maturation products of the SMR1 protein, specifically a peptide of structural formula XQHNPR (SEQ ID NO:14) recognize specific target sites in organs that are deeply involved in the mineral ion concentration. This discovery has led the inventors to assign to the SMR1 pentapeptide, hexapeptide or undecapeptide an active role in the regulation of the metal ion concentrations in the body fluids and tissues, and thus a therapeutic role of these peptides in all the metabolic disorders related to a mineral ion imbalance.
Thus, the present invention concerns the therapeutic use of the peptide of structural formula XQHNPR (SEQ ID NO:14) wherein X denotes a hydrogen atom or X represents an amino acid chain chosen from the following: X=V (SEQ ID NO:5) or X=VR (SEQ ID NO:5) or X=VRG (SEQ ID NO:5) or X=VRGP (SEQ ID NO:5) or X=VRGPR (residues 1-5, SEQ ID NO:7) or X=VRGPRR (residues 1-6, SEQ ID NO:7), for preventing or treating diseases caused by a mineral ion imbalance in a mammal, specifically in human.
More particularly, one object of the present invention is the use of the above-described therapeutic peptides for treating bone, teeth, renal, kidney, intestine, pancreas, stomach mucosa or parathyroid disorders caused principally by a mineral ion imbalance in the body fluids or tissues.
Accordingly, the therapeutic peptides according to the present invention are used for preventing or treating diseases like hyper- or hypo-parathyroidism osteoporosis, pancreatitis, submandibular gland lithiasis, nephrolithiasis or osteodystrophy.
The discovery of the therapeutic activity of the SMR1 protein and its derived peptides as well as their physiological in vivo targets has allowed the inventors to design new molecules that can be considered as biologically active derivatives of the above-described therapeutic peptides.
Such biologically active derivatives of the therapeutic peptides according to the invention are peptides that are structurally and chemically related to the XQHNPR (SEQ ID NO:14). such as peptides having the same amino acid sequence as the initial peptides but containing one or several modified amino acids that are able to confer a better in vivo stability to the therapeutically active molecule and which possess the same biological activity as the endogenous peptide or which behave as an antagonist molecule of the endogenous peptide.
Thus, the therapeutic use of peptides that are homologous to the XQHNPR (SEQ ID NO:14) peptide is also part of the present invention. By homologous peptide according to the present invention is meant a peptide containing one or several amino acid substitutions in the XQHNPR (SEQ ID NO:14) sequence. The amino acid substitution consists in the replacement of one or several-consecutive or non-consecutive-amino acids by xe2x80x9cequivalentxe2x80x9d amino acids. The expression xe2x80x9cequivalentxe2x80x9d amino acid is used herein to name any amino acid that may substituted for one of the amino acids belonging to the initial peptide structure without modifying the hydrophilicity properties and the biological target of the initial peptide structure. Preferably, the peptides containing one or several xe2x80x9cequivalentxe2x80x9d amino acids retain their specificity and affinity properties to the biological targets of the XQHNPR (SEQ ID NO:14) peptide. In other words, the xe2x80x9cequivalentxe2x80x9d amino acids are those which allow the generation or the obtention of a polypeptide or peptide with a modified sequence as regards to XQHNPR (SEQ ID NO:14), said modified polypeptide or peptide being able to act as an agonist or an antagonist molecule of the XQHNPR (SEQ ID NO:14) peptide.
These equivalent amino acids may be determined by their structural homology with the initial amino acids to be replaced and by their biological activity on the target cells of the XQHNPR (SEQ ID NO:14) peptide.
As an illustrative example, it should be mentioned the possibility of carrying out substitutions like, for example, leucine by valine or isoleucine, aspartic acid by glutamic acid, glutamine by asparagine, arginine by lysine etc., it being understood that the reverse substitutions are permitted in the same conditions.
By modified amino acid according to the present invention is also meant the replacement of a residue in the L-form by a residue in the D form or the replacement of the glutamine (Q) residue by a Pyro-glutamic acid compound. The synthesis of peptides containing at least one residue in the D-form is, for example, described by Koch et al. in 1977.